This project is designed to explore pathways and determinants of cell death induced by photodynamic therapy (PDT), a form of cancer treatment that circumvents most drug-resistance mechanisms. An understanding of death pathways is expected to increase our knowledge of factors in PDT efficacy (cells killed per photon absorbed). PDT can induce an apoptotic response if the cell phenotype permits. If apoptosis is impaired, another death pathway will be utilized;this doubtless contributes to PDT successes. In an exploration of alternative pathways, we found substantial evidence that autophagy accompanies photodamage as cells attempt to survive, but can also be a death pathway if apoptosis is impaired. Autophagy can promote survival by the internal digestion and recycling of cellular components, and by eliminating damaged organelles. In the contect of PDT, we propose that autophagy can either enhance survival or be a pathway to cell death. The latter appears to predominate when the cell phenotype prevents apoptosis. We plan to examine death pathways as a function of cell type (carcinoma, leukemia/ lymphoma, non-malignant) and the PDT target. The Bcl-2 antagonist HA14-1 will be utilized to assess consequences of Bcl-2 inactivation since this protein is often a PDT target and its loss can trigger both apoptosis and auto- phagy. Results will be correlated with PDT dose, photodamage sites and clonogenic viability studies. Since many proteins involved in the autophagic program are membrane-bound, a search for photodamage to such proteins is considered important. Methods for assessing apoptosis are well-defined, and involve detection of caspase activation, DNA fragmentation to endonucleosomes and chromatin condensation. Autophagy is currently characterized by formation of double-membrane intracellular vesicles containing cellular organelles and cytosol, and processing of the Atg8 ortholog LC3. Experiments are planned to assess the degree of protection from photokilling offered by autophagy and the ultimate nature of the cell death that initially involves extensive cell vacuolization and LC3 processing. Effects of PDT targets and a sub-contract with LSU will provide a synthetic capacity for preparation of photosensitizers and inhibitors that are not commercially available. Significance: The ability of PDT to mediate direct photokilling of tumor cells, e.g., at surgical margins, can be an important aid in tumor eradication. Information on mechanisms of cell death pathways and their role in cancer control is also expected to assist in drug development and to provide better understanding of the role of death pathways in cancer control.